17
Immunological Diagnosis
of Active and Latent TB
Beata Shiratori, Hiroki Saitoh, Umme Ruman Siddiqi,
Jingge Zhao, Haorile Chagan-Yasutan, Motoki Usuzawa,
Chie Nakajima1, Yasuhiko Suzuki1 and Toshio Hattori
1Hokkaido
Tohoku University, Graduate School of Medicine
University, Research Center for Zoonosis Control
Japan
1. Introduction
Immunological diagnosis of tuberculosis (TB) is based on the immune responses against
Mycobacterium tuberculosis (MTB). Immunological diagnosis can detect both active and
latent TB, and can detect not only pulmonary TB but also extra-pulmonary TB. Compared to
conventional diagnosis, immunological diagnostic tests have eminent advantages. On the
other hand, there are still some limitations. As is known, various mycobacteria share
homologous proteins, that lead to immunological cross reaction. To correctly detect TB
infection, we need to choose a method that initiates the anti-MTB immune response
properly. Human immunodeficiency virus (HIV) infection weakens the immune response,
which may lead to false-negative results. HIV infection accompanied by TB is another
urgent issue in global health. In this chapter, we will explain the immunological responses
to MTB and the immunological interaction between HIV and TB. We will then introduce
each diagnosis from the immunological point of view, and describe novel assays which we
are now developing.
2. Immunological response to MTB
In this section, we describe the immune response to MTB and MTB/HIV co-infection.
2.1 Innate immunity and adaptive immunity
When the immune system encounters foreign organisms, it works to eliminate them and
both innate immunity and adaptive immunity are engaged in this process (Murphy, 2011).
In this section, we will briefly describe the immune responses to give the theoretical basis
for the immunological diagnosis of TB.
Innate immunity is a non-specific response to pathogen and is the first line of defence
against microorganisms. When macrophages recognize foreign organisms, the cells ingest
and digest them. Receptors on the cell surface, especially those of toll-like receptor family,
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are involved in this process. Then macrophages express digested fragments on their surface
promoting the initiation of the secondary response, the adaptive immunity.
Adaptive immunity is antigen specific and creates immunological memory. Responding T
cells are functionally divided into T-helper cells 1 (Th1) and T-helper cells 2 (Th2), which are
activated by antigen presentation through major histocompatibility complex class II (MHC
II) on macrophages. Th1 cells stimulate T cell populations that secrete interferon gamma
(IFN-) and interleukin-2 (IL-2) to activate cytotoxic T cells and, finally, to eliminate foreign
organisms. This response is known as cell mediated immunity. Th2 cells secrete interleukin4 (IL-4) to stimulate B cells, which produce antibody against the pathogen. This response is
known as humoral immunity. Immunological diagnosis is based on the adaptive immune
response to a targeted pathogen. If we detect or measure the activation of the immune
response induced by MTB, we can diagnose MTB infection.
Recently, Th17 cells, which are responsible for inflammation, and T regulatory (Treg)
populations, which suppress a variety of immune responses, have received much attention.
2.2 Immunological responses to MTB
MTB enters our body through the airway in droplet nuclei and is phagocytosed by alveolar
macrophages. Macrophages digest MTB to connect MHC II molecules in the cell and
fragments of MTB, then present their complex on the cell surface. Antigen presentation
generates adaptive immunity(Walzl et al., 2011).
Th1 cells are activated and produce cytokines, such as IFN- and IL-2. These cytokines
activate cytotoxic T cells and macrophages. IFN- enhances the anti-microbial activity of
macrophages.
Th2 cells, producing IL-4, promote TB specific antibody production by B cells. These
interactions lead to the generation of memory cells. There are two main populations of
memory cells, effector memory T cells (which may be transiently present in the blood if
bacteria are cleared) and central memory T cells (which may remain for life but may not
provide protection in all individuals). The recently developed IGRA test measures IFN-
produced by effector memory T cells (Horsburgh & Rubin, 2011).
Despite these sophiscated immune responses, they often fail to eliminate MTB from the
body and the bacteria may exist in a quiescent state for a prolonged period. Such a state is
called latent tuberculosis infection (LTBI). In 10% of infected individuals, active TB develops
and more than 80% of new cases of TB result from reactivation of the primary infection. The
increase of HIV rates facilitates the reactivation of TB due to the imunosuppression.
2.3 TB with HIV infection
This lethal combination of TB/HIV is anything but rare; demographic analyses have
estimated that over 60 % of the population in the sub-Saharan region has been infected by
MTB, which has become a leading cause of mortality among HIV patients. At the end of
2009, TB infection was reported to be responsible for 13% of HIV deaths (Science Daily,
2009). Retrospective studies concluded that, among HIV/TB patients, 7% to 45% of them
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probably develop symptoms homogeneous with Immune Reconstitution Inflammatory
Syndrome (IRIS) (Murdoch et al., 2007; Shelburne et al., 2005). Therefore, exploring the
relationship between TB and HIV become necessary.
Since HIV virus can weaken the immune system, LTBI can be activated resulting in
pulmonary or extrapulmonary TB. A variety of immune cells and immune cytokines are
involved in the reactivation of LTBI. Some cytokines, such as interleukin-8 (IL-8) and
interleukin-12β (IL-12β), may be used as biomarkers to monitor the immune reaction to
LTBI (Wu et al., 2007) and possibly shed light on preventing of LTBI progression in HIV
patients (Walzl et al., 2011). So far, various biomarkers to characterize LTBI and TB in HIV
patients have been proposed. Without medical intervention, HIV infection will progress to
acquired immune deficiency syndrome (AIDS), accompanied by multi-microbial infections,
including TB infection, which often proves lethal.
2.3.1 Mechanism of immunological interaction of HIV and TB
Infection with HIV enhances the susceptibility to MTB infection. Because the occurrence of
these two diseases is heavily dependent on the immune system, their interactions are more
complex than previously understood. Previously, we studied the plasma levels of two
matricelluar proteins such as galectin-9 (GAL-9) and osteopontin (OPN) in AIDS patients
complicated with various opprotunistic infections (Chagan-Yasutan et al., 2009). The levels of
both molecules were high in all the patients but only the level of GAL-9 decreased and that of
OPN remained high after Highly Active Antiretroviral Therapy (HAART). Also, As Figure 1
shows, it was noted that the GAL-9 level was exceptionally high in acute HIV infected
individuals (Chagan-Yasutan et al., 2009). The cellular receptor for GAL-9 is the T-cell
immunoglobulin domain and mucin domain 3 (Tim-3) and Tim-3 is expressed on Th1 cells.
Mycobacterium tuberculosis-infected macrophages express GAL-9 and the Tim-3 GAL-9
interaction leads to macrophage activation and stimulates the bactericidal activity by inducing
caspase-1–dependent interleukin-1β (IL-1β) secretion. Therefore, Th1 cell surface molecule Tim-3
may have evolved to inhibit the growth of intracellular pathogens via its ligand GAL-9, which is
also known to inhibit the expansion of effector Th1 cells (Jayaraman et al., 2010). Therefore, only
one case of MTB associated with acute HIV was reported (Crowley et al., 2011).
In contrast, chronic HIV infected individuals succumb to various opportunistic infections
and pulmonary TB is known to occur when CD4+ T cell numbers are still high, indicating
that the immune system plays a role in the development of pulmonary TB (Holmes et al.,
2005). Similarly, T cell epitopes of different strains representative of global diversity are
highly conserved in MTB (Comas et al., 2010). Due to the conserved epitopes, the host can
maintain MTB for a long time as latent infection and can transmit it to the next generation. It
is also suspected that CD4+ T cells have an essential role in tissue damage that results in
cavity formation, which enhances aerosol infection. In HIV endemic areas, the situation
becomes more complex if the CD4+ T cells numbers increase after HAART and then
recovery of the immune system is variable (Fig 2).
2.3.2 Network among LTBI and HIV
A. Healthy Individuals. For immunologically potent people, the reaction to invading
microbes consists of innate and adaptive immune mechanisms. Genome research has
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Fig. 1. Proposed biological effects of GAL-9 in HIV or HIV/TB infection.
The plasma levels of GAL-9 were elevated in chronic AIDS patients as well as in TB patients
(non-published data), but were exceptionally high in acute HIV infection (Chagan-Yasutan
et al., 2009). It was reported that GAL-9 interacts with its Tim-3 ligand to regulate the
overexpansion of Th1 cells to induce apoptosis (Zhu et al., 2005; Kashio et al., 2003). In TB,
however it was speculated that GAL-9 contributes to the activation of macrophage cells
(Mø) and then inhibits intracellular bacterial infection by caspase-1 dependent IL-1β
production (Jayaraman et al., 2010).
revealed that TB epitopes binding to human CD4 + T cells are conserved (Comas et al.,
2010). Accordingly, during the long history of fighting against TB, human beings have
evolved a spectrum of potential TB-specific naive CD4+ T cells, which can be activated as
soon as TB invades and transformed to TB effective CD4+ T cells to fight against TB bacilli,
some of which would transform into central memory T cells (CMT) after TB is controlled.
Such CMT cells are capable of quick proliferation once they confront TB. During infection,
these TB antigen specific CD4 + T cells make up a certain percentage of CD4+ T cells; the
percentage could be affected by the volume of CMT and individual differences in gene
expression profiles (Maertzdorf et al., 2011).
B. LTBI (Latent tuberculosis infection) Individuals. Thirty percents of the population in the
world is infected by MTB. However, effector CD4+ T cells protect LTBI individuals from
developing active TB. In this case CMT act as a backup to proliferate of effector CD4+ T
cells. Observation of this TB-specific reaction can be simplified by monitoring the IFN- level
in IGRA (Rueda et al., 2010). Apart from IFN-, other biomarkers including CD154 and
CD107 also indicate a TB-specific reaction (Streitz et al., 2011). IFN- has multiple effects on
the immune system to control TB. As IFN- is secreted by TB effector CD4+ Th1 cells, it
could mediate cytotoxic T cells to recognize and damage TB-infected macrophage cells. IFN-
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Immunological Diagnosis of Active and Latent TB
357
 also enhances MHC II on macrophages. Other cytokines such as IL-2 and IL-4 could act in
synergy with IFN- to control LTBI. However, 10% of them eventually progress to active TB
(Day et al., 2011).
Fig. 2. Schematic network among LTBI and HIV.
The horizontal plane indicates the make up of inflammatory CD4+ cells and TB effective
CD4 + T cells (Effector memory T cells), which are coloured in blue and yellow, respectively.
The overall reaction of different cells to TB is described according to the volumes of the
different colours, the vertical lengths of which indicate biomarkers that rise up or descend
resulting from TB reaction. Such schema and many exceptional cases are present in
HIV/AIDS and tuberculosis.
C. Pulmonary TB. A pulmonary cavity is a typical sign of pulmonary TB. A recent paper
reported that CD4+ T cells could be an essential factor for TB cavity formation (Russell et al.,
2010). Patients of pulmonary TB have been reported to have mainstream TNF- TB specific
CD4+ T cells, which can lead to an inflammatory reaction (Indicated by blue colour in
vertical direction of C) in active TB patients. A recent study based on 101 TB and LTBI
individuals described that TNF- specific CD4+ T cells might be an important biomarker for
diagnosing active TB. In the majority of active TB patients TNF- can be detected in the
MTB antigen stimulated cells. Flow-cytometry showed that inflammatory-related CD4+ T
cells represent 37.4% of total CD4+ T cells (indicated by area between blue and yellow
colour in C) is the cut-off of LTBI becoming active TB (Harari et al., 2011). And such TNF-
TB specific CD4+ T cells activate other inflammatory cells. However, IFN-inducible
neutrophils may also play an essential role in eliciting inflammatory CD4+ T cells.
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Neutrophils corresponding to IFN-, and  modulate an inflammatory reaction in active
TB patients (Berry et al., 2010).
D. Extrapulmonary TB. Due to the small number of inflammatory CD4+ T cells, TB lacks the
ability to form cavities in the lungs and lead to multi-organ or systemic infection. Such
patients are frequently found among those with AIDS and patients on immunosuppressive
therapy. Interestingly, extrapulmonary TB is a prominent risk factor for IRIS. (Manosuthi et
al., 2006)
E. The stage between LTBI and active TB infection. It features opacities in the lungs, but
sputum and IGRAs test are negative.
F1, G1. HIV-infected Patients. In Sub-Saharan region, frequently seen cases are LTBI
individuals infected by HIV. Asymptomatic HIV can last by 2 years to 10 years in normal
individuals after infection. As a result of HIV infection, CD4+ cells drop progressively. After
HIV infection, the virus targets all CD4+ T cells including effector, inflammatory and CMT
CD4+ T cells and anti-HIV drugs restores their function. F1 indicates those patients who
didn’t carry larger number of CMT cells and could not produce enough effector CD4+ T
cells after TB stimulation. G1 indicates that those patients with a large pool of TB CMTs or
who has been infected by LTBI prior to HIV infection and then carried more LTBI
stimulating specific effector CD4+ T cells or memory T cells (Mueller et al., 2008). F1, G1 will
finally process to extrapulmonary TB (indicated by E), if no medication intervenes.
F1-F2. HIV Patients during HAART treatment. In macaque experiment, SHIV was found to
preferentially infect CMT cells (He et al., 2011). When HAART treatment is applied, CD4+ T
cells count will rise. Occasionally, such rise causes IRIS, because CMT cell count will bounce
up and effector CD4+ T cells will show increased activity. However MTB specific
inflammatory CD4+ T cells will dominate their large number. (Indicated by proportional
volume between blue and yellow). As TB Inflammatory CD4+ T cells lead the reaction,
patients have similar prognoses as pulmonary TB (Indicated by C) (Worodria et al., 2011).
G1-G2. HIV Patients after HAART treatment. HAART will rapidly reconstitute the immune
surveillance (indicated by elevation of yellow column) (Hua et al., 2011). The occurrence of
TB therefore decreases (Sant'Anna et al., 2009).
Net work
C-F1-F2. TB and HIV stimulate specific inflammatory T cells to produce TNF- which, in
turn, help them to progress at faster rate (Sorathiya et al., 2010). The patients can be treated
by anti-TB therapy followed by HAART. Occasionally, paradoxical TB IRIS1 occurs,
probably caused by MTB specific inflammatory CD4+ T cells. MCP-1 (monocyte chemotactic
protein 1) was found to be a reliable candidate biomarker to screen patients who may
develop to paradoxical IRIS (Haddow et al., 2011). B-G1-G2. HAART strengthen immune
responses against MTB and can decrease the occurrence of TB (Middelkoop et al., 2011, Hua
1 Definition of TB IRIS: ‘paradoxical’ worsening of symptoms of known disease, either at a new body
site or at the original body site, with an incidence of 8–43% of TB-co-infected individuals starting ART;
or ‘unmasking’ of occult Mycobacterium tuberculosis infection, in which infection was not clinically
apparent prior to ART but presents floridly during ART, affecting around 5% among those starting ART
without known TB infection in South Africa.
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Immunological Diagnosis of Active and Latent TB
et al., 2011). Notably, HAART unmasking TB manifestation can be found in some cases and
C-reactive protein (CRP) was reported to be helpful to detect the development of unmasking
TB-IRIS cases (Haddow et al., 2011).
3. Immunological diagnosis
Over decades, there have been attempts to find new diagnostic tools, that are sensitive and
specific, simple, inexpensive and able to distinguish latent tuberculosis from active
tuberculosis as well as MTB infected individuals from uninfected ones.
3.1 Tuberculin skin test
Tuberculin skin test, also called as Mantoux skin test, has been used for the diagnosis of
tuberculosis for more than a century. Despite the numbers of logistic and performance
problems and poor specificity, TST is still performed as a routine diagnostic method. The
purified protein derivative (PPD) antigens, that are used for TST are highly homologous to
antigens of Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine and nontuberculosis mycobacteria (NTM) antigens. These and other factors may lead to falsepositive or false negative TST results.
Although other antigens have been evaluated as a skin test reagents e.g.molybdopterin-64
(MPT-64) and molybdopterin-59 (MPT-59), none of them proved superior to tuberculin skin
test (Wilcke et al., 1996).
False-positive reactions

Infection with NTM

Previous BCG vaccination

Incorrect method of TST
administration

Incorrect interpretation of reaction

Incorrect bottle of antigen used
False-negative reactions

Cutaneus anergy

Recent TB infection

Very old TB infection

Very young age (less than 6 months
old)

Recent live-virus vaccination (e.g.,
measles and chicken pox)

Incorrect method of TST
administration

Incorrect interpretation of reaction
Table 1. False-positive and false-negative TST reactions. (CDC, 2010)
3.2 Interferon Gamma Releasing Assay (IGRA)
Because IFN- is a cytokine that plays a critical role in resistance to Mycobacterium
tuberculosis infection and MTB infected individuals respond to MTB antigen stimulation by
releasing increased amounts of this cytokine from effector memory cells, methods based on
measuring the IFN- production by antigen stimulated human T lymphocytes have been
developed. There are two new blood tests on the market, the enzyme-linked immunospot
assay (ELISpot) (T-SPOT®.TB, Oxford Immunotec, Oxford, UK) and the enzyme-linked
immunosorbent assay (ELISA) (QuantiFERON-TB Gold In-Tube, QFT-GIT, Cellestis,
Carnegie, Australia). Both IGRAs have high sensitivity and specificity, for QFT-GIT 81%-
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92.6% and 98.8%-99.2% and for T-SPOT®.TB 87.5%-95.6% and 86.3%-99.9%, respectively
(Diel et al., 2008; Harada et al., 2008; Oxford Immunotec, 2011). Firstly, it should be
mentioned that using IGRA it is impossible to distinguish between latent and active TB for
which no such a method yet exists. However the detection of both latent and active TB has
been markedly improved by employing IGRA methods. The factor, that increased the
sensitivity and specificity of IGRA was the discovery and use of antigens encoded by
Regions of Difference 1 (RD1) in the MTB genome, which is absent in BCG vaccination or
NTB. Among the nine antigens encoded by RD1, early secreted antigenic target 6kDa
(ESAT-6) and culture filtrate protein 10kDa (CFP-10) are used as a stimulatory antigens.
However, ESAT-6 and CFP-10 antigens are also present in NTM, namely M. leprae, wild
type M. bovis, M. marium, M. kansasii, M. sulgai, M. flavescens, in NTM endemic areas,
IGRA results might be false positive and make it difficult to distinguish MTB between NTM.
3.2.1 QuantiFERON-TB Gold In Tube assay
In comparison to the previous form of QuantiFERON Gold, the QuantiFERON Gold In Tube
(QFT-GIT) version enables immediate antigen stimulation of lymphocytes within whole
blood. In addition to ESAT-6 and CFP-10, QFT-GIT contains a peptide from the internal
section of TB 7.7 (Rv2654), which may increase the sensitivity of the test (Aagaard et al. 2004;
Brock et al., 2004), though it is arguable whether specificity is improved. All three antigens
are present on the wall surface of the blood collection tubes. Besides the immunity status
and the absolute and relative lymphocyte number there are external factors that may
influence the QFT results, e.g. drawing an adequate volume of blood, appropriate
attachment of lymphocytes to the antigens, sample handling and the ELISA assay
procedure. Another important issue is the interpretation of the results. There are two cutoffs given by the manufacture, 0.35 IU/l and 0.1 IU/l. A value of more than 0.35 IU/l seems
to be appropriate for good discrimination of truly infected individuals (Harada et al., 2008).
On the other hand, the values between 0.35 IU/l and 0.1 IU/l, the intermediate result,
should take into account the individual patient`s condition (Harada et al., 2008; Liote H &
Liote F, 2011). In Japan, the interpretation criteria differ from those anywhere else.
Intermediate results in Japanese people are suspected to be positive and are flagged for
follow-up observation (Prevention Committee, Japanese Society of Tuberculosis, 2006).
Depending on the MTB infection prevalence, it has been suggested to use different cut-offs
(Harada et al., 2008). In areas where the MTB infection prevalence is low, the specificity is
probably of great importance. However, in high-risk TB screening situations, identification
of LTBI is likely to be more important than the potential side effects of the MTB treatment
and a cut-off value of 0.1 IU/l should be employed (Yew & Leung, 2006).
®
3.2.2 T-SPOT .TB test (ELISpot)
Using the ELISpot assay it is possible to visualize and count MTB-specific memory T cells
producing IFN-. The great advantage of this test is that each test well contains the same
number of peripheral blood mononuclear cells (PBMCs). Especially in the patients with low
T cell counts from HIV or other immune disorders, it enables the objective evaluation by
adjusting the designed cell number. While in QFT-GIT all antigens are present in the same
tube, in the ELISpot assay ESAT-6 and CFP-10 antigens are added and read separately. The
manufacture claims that this assay has very low cross-reactivity with NTM (Oxford
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Immunotec, 2011). The important step is the accurate adjustment of the cell count. However,
if the cells are not well adjusted and the nil control contains more spots than indicated in the
instructions, the test needs to be repeated. What is more, to run the ELISpot assay, a trained
professional should be engaged and other special tools such as a plate reader are needed.
Exact interpretation of the results is crucial, so the manufacturer prepared a training manual
for distinguishing between valid and invalid spots. The ELISpot assay`s intermediate result
rate is 3-4% (Dosanjh et al., 2008), which is significantly lower than that for QFT assays (1121%) (Ferrara et al., 2006; Piana et al., 2006).
QFT-GIT test has recently become routinely used as a diagnostic tool for MTB, but the TSPOT®.TB test is employed only in few countries, mostly because of the high cost of this
assay. In conclusion, IGRA seems to be beneficial tool for TB diagnosis, especially for people
with a high-risk of developing active TB.
3.2.3 Other approaches of LTB diagnosis
In addition to ESAT-6 and CFP-10, other antigens have been proposed for ELISpot assay but
have not been implemented for commercial use. Addition of the novel antigen Rv3879c
increased the diagnostic sensitivity of the standard ELISpot assay and, in combination with
TST, reached a sensitivity of 99% (Dosanjh et al., 2008). Other researchers showed that
antigen heparin-binding-hemagglutinin was significantly more sensitive than ESAT-6 and
more specific than PPD for the detection of LTBI (Hougardy et al., 2007a).
3.2.4 Conditions altering IGRA results
There are various conditions such as oncologic disease, HIV infection, anti-TNF-, corticoid
or other immunomodulatory therapy, diabetes mellitus, renal failure or other
immunocompromising conditions that are responsible for intermediate or false negative
results (Schoepfer et al., 2008; Matulis et al., 2008; Kim et al., 2009). Particularly, the antiTNF- treatment has increased recently, which hampers the activation of the innate immune
responses, T cell mediated adaptive immune response and production of protective IFN-.
Similarly, metabolic diseases such as diabetes mellitus are known to affect chemotaxis,
phagocytosis, activation, and antigen presentation by phagocytes in response to MTB, and
this defect does not improve with insulin (Moutschen et al., 1992). Importantly, IFN-
production was found to be impared in hyperglycemia (Yamashiro et al., 2005), but another
study showed that both IGRA results were not affected in diabetes mellitus patients (Walsh
et al., 2011). Intermediate results have been found to be also associated with lower serum
albumin and double immunomodulatory therapy (Papay et al.,2011).
3.3 Role of regulatory T cells in diagnosis of MTB
Since TB is a chronic disease in which bacilli evade the immune system to persist in the host
organism, scientists are trying to find and understand the mechanism of MTB
immunopathology. It is still unclear what conditions prone to MTB infection, what factors
are involved in TB latency, activation or masking of the disease and what causes the
imbalance of the immune responses that finally lead to the failure of MTB eradication. The
immune system posses a regulatory mechanism in which Treg cells play essential roles in
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establishing and sustaining self tolerance and immune homeostasis as well as regulate the
host response to infection.
3.3.1 Role of Treg in mycobacterium tuberculosis infection
The first demonstration of the suppressive capacity of T cells was performed in 1973 in an
animal model of bacillus BCG vaccination. Thymocytes from BCG-injectected rats were
harvested and transfected to alive normal recipients. Subsequently, recipients were
challenged with the same antigen and the inhibition of their skin reaction was observed (Ha
& Waksman, 1973).
The number of CD4+CD25+FoxP3+ Treg cells was found to be increased in the blood or at
the site of infection in active tuberculosis patients (Guyot-Revol et al., 2005) and the
frequency of CD4+CD25+FoxP3+ T lymphocytes was inversely collated with the local MTBspecific immunity, and both blood and pleural Treg cells were able to suppress IFN- and
IL-10 production in TB patients (Chen et al., 2007). This mechanism is thought to contribute
to the pathogenesis of human TB (Guyot-Revol et al., 2005; Chen et al., 2007). Treg cell
expansion is believed to predispose or be a marker of the progression of latent TB to active
disease (Hougardy et al., 2007). What is more, it was found that depletion of CD4+CD25+ T
cells enhanced the protective IFN- production in TB patients (Guyot-Revol et al., 2005) and
transiently reduced the bacterial load and granuloma formation (Ozeki et al., 2010).
3.3.2 Objectives of our Treg study
The majority of individuals vaccinated with BCG or infected with MTB develop a delayedtype hypersensitivity which is manifested as a positive response of intradermal injection to a
purified protein derivative from MTB. But about 15% of active TB patients show false
negative results and are considered to be anergic TB (Bloom & Small,1998). Similarly, in HIV
infected individuals, TST results are often found to be false-negative. Concerning the high
frequencies of Treg cells in both diseases (Chen et al., 2007; Bi et al., 2009), it is highly
probable that Treg cells may play central role in the anergy mechanism. Cutaneus anergy in
active TB is associated with the absence of granuloma formation and poor clinical outcome
(Boussiotis et al., 2000). Another study showed that, in an anergic patient, sustained MTB
stimulation led to enhanced IL-10 production and the generation of anergic MTB-specific T
regulatory cells with the Tr-1 phenotype (Boussitis, 2000). In certain closed populations, a
high percentage of TB anergy and high prevalence of active TB were observed, which led to
the speculation that innate genetic factors may play a role (Delgado & Ganea, 2001). We
observed several cases of anergy in health care workers (HCW), who had been in close
contact with active TB patients. Therefore, we questioned whether Treg cell may mask latent
TB infection.
3.3.3 Materials and methods
Human subjects and samples
According to previously obtained TST results, one TST positive (27 years old) and one TST
negative (42 years old) healthy (X-ray and sputum smear negative) health care individuals
with no history of previous MTB infection or other chronic disease were recruited in this
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study. A TST result ≥ 10 mm was considered to be positive. Both individuals were
vaccinated with BCG when young. The protocol was approved by the ethical commitee of
Tohoku University Medical School. Written informed consent was obtained. Both
individuals were HIV, hepatitis B and C virus negative.
From each donor 20 ml of Ethylenediamine tetraacetic acid (EDTA) treated peripheral
venous blood was obtained, centrifuged and the peripheral blood mononuclear cells
(PBMCs) were isolated by Ficoll separation (Ficoll-Paque PLUS,GE Healthcare Bio-Science
AB, Uppsala, Sweden). After washing twice in Phosphate Buffered Saline (PBS), PBMCs
were resuspended in complete RPMI 1640 medium (consisting of RPMI 1640 supplemented
with 10% heat-inactivated fetal calf serum, 2% glutamine and 1% penicilin/streptomycin) at
a concentration 5x106 cells/ml.
Depletion of CD4+CD25+ cells
The cells designed for depletion were centrifuged and resusupended in Running buffer
(MACS Separation Buffer, Miltenyi Biotec). To avoid unspecific binding of the antibody, 100
ul of FcR Blocking Reagent (Miltenyi Biotec) was added to the PBMCs which were then
incubated for 15 minutes on ice. After adding 25 ul of CD25-Biotin monoclonal antibody
(Miltenyi Biotec) and 15 minutes incubation on ice, the cells were washed with Running
buffer. To cells resuspended in Running buffer, 25 ul of anti-Biotin Microbeads (Miltenyi
Biotec) were added, followed by incubation for 15 minutes on ice. Subsequently, the cells
were washed and resuspended in Rising Solution (Miltenyi Biotec) and CD4+CD25+ T cells
were depleted by positive selection using a magnetic-activated cell sorter (MACS)-assisted
cell sorting system (Miltenyi Biotec, Auburn, CA) according to the manufacturer´s
instructions.
Cell phenotype determination
To visualize the biotinylated mAb on CD25+ cells, streptavidin-allophycocyanin (BD
PharMingen) was used and the CD4+ cells were stained by anti-CD4 FITC antibody. Flow
cytometric analyses were performed with a FACSCalibur flow cytometer (Becton Dickinson)
using the CELL quest program (Becton Dickinson). More than 90% of CD4+CD25+ cells
were depleted from the PMBCs of both individuals.
Cultivation and cytokine determination
Freshly isolated, undepleted and Treg depleted PBMCs were cultured in triplicate in 96-well
plates at a concentration of 2x105 cells per well in 200 ul of complete RPMI 1640 medium
and incubated for 24 hours at 37 °C with 5% CO2. The cells were stimulated with 1 ug/ml of
PPD (Statens Serum Institute, Copenhagen, Denmark), 500nM of recombinant CFP-10 and
ESAT-6 protein antigen. The cell culture supernatant was harvested from each well for
ELISA analysis. IFN-gamma and IL-10 production was determined by using human IFNgamma and IL-10 BD Opt EIATM Set according to the manufacturer´s instructions (BD
Bioscience). Optical densities were read at 450 nm on an ELISA plate reader (VersaMax-KT,
Molecular Devices Corp., CA, USA ) and the concentrations were calculated from standard
curves using the Soft Max Pro program (Molecular Devices Corp.).
Statistical analysis
Two-sided paired t-test was used to analyze the effect of Treg on cytokine production.
Differences were considered significant when the p value was less than 0.05.
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3.3.4 Results
To investigate the immunosuppressive effect of Treg cells on the anti-TB immune response,
CD25+ T cells were depleted from PBMCs of TST positive and TST negative healthy HCW.
IFN- and IL-10 production upon PPD, CFP-10 and ESAT-6 stimulation was assayed.
The positive response to PPD stimulation in the TST-positive individual agreed with the
TST result. Similarly, the low IFN- response to PPD in the TST anergic person supported
the unresponsiveness of PBMCs to PPD stimulation. Both individuals had low levels of IFN on CFP-10 stimulation, but the TST anergic person, in contrast to the TST positive one,
responded to ESAT-6 stimulation, what is suspectious for LTBI. Depletion of Treg cells
significantly enhanced the IFN- production in the TST anergic person, but in the TST
positive person it influenced the IFN- level only after PPD stimulation (Fig. 3A). Depletion
of Treg cells significantly influenced the IL-10 production only by PPD and CFP-10, but not
by ESAT-6 stimulation (Fig. 3B).
3.3.5 Discussion
PPD is the prototypical mycobacterial antigen, which is included also in the Mantoux skin
test antigens. The result with PPD stimulation demonstrated a T cell antigen recognition
level consistent with TST results. PPD unresponsiveness in anergic TB patients might be due
to the inability of their antigen presenting cells to present antigens or to the inability of their
T cells to respond to antigen-specific stimulation (Boussiotis et al., 2000). Our results showed
that PPD anergy might be due to an impaired T cell response as an effect of the Treg cell
activity.
Recombinant antigens such as ESAT-6 and CFP-10 have been reported to be strong IFN-
inducers. CFP-10 was reported to be less reactive in comparison to ESAT-6 in TB patients as
well as in healthy controls (Oliveira et al., 2007), which was also observed in our assay. The
anergic subject in our study showed a strong response to ESAT-6 stimulation, which made
him highly suspected to be TB infected.
CD4+CD25+FoxP3+ T cells have been found to be increased in MTB infection and to
suppress the MTB-specific immunity (Hourgady et al., 2007; Chen et al., 2007). It has been
found that elimination of CD4+CD25+ T cells significantly increased the BCG-induced
production of IFN- and IL-10 by PBMCs from patients with active TB, but not by those
from healthy volunteers (Chen et al. 2007), but there is no report of Treg functions in TST
anergic LTBI. We have demonstrated for the first time that Treg cell depletion in anergic
individual led to enhanced IFN- production upon MTB specific ESAT-6 and CFP-10 antigen
stimulation, while in the TST positive healthy individual we did not observed such a
phenomenon. These results support the argument that CD4+CD25+ T cells suppress the Th1
immune response.
IL-10 is considered a soluble factor that plays a central role in controlling inflammatory
processes, suppressing T cell responses, and maintaining immunological tolerance (Moore et
al., 2001). In the condition of MTB infection, mycobacteria-induced IL-10 production by
macrophages allow mycobacteria-infected cells to elude immune surveillance (Larsen et al.,
2007).
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Immunological Diagnosis of Active and Latent TB
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Fig. 3. Immunossuppresive effect of Treg on (A) IFN- production and (B) IL-10 production.
Statistically significant (*) p < 0.05
Antigen presenting cells matured in the presence of BCG are able to instruct naive T cells
to develop into cytokine-producing T cells that can be categorized into Th1 (IFN-
producing), Th2 (IL-4-producing) or Tr1 (IL-10-producing) cells (Larsen et al. 2007). We
can speculate that upon first contact with BCG vaccine in early childhood, the naive T cell
development may lead to polarization of the immune response in favor of Th2 and Tr1 IL10-producing cells. An anergic individual, in comparison to a TST positive person, have
elevated levels of IL-10 upon MTB antigen stimulation and it might be possible that his
immune responses are switched towards an IL-10 immune response. The anergizing effect
and antiinflammatory properties of IL-10 might be one of the factors maintaining the
anergy and LTBI chronicity.
Even if this is a sporadic finding, we believe that these results enable a better understanding
of the immune mechanisms involved in anergy and LTBI in adult, healthy, BCG-vaccinated
individuals. It is necessary to confirm these data in larger numbers of volunteers. It might be
disputed whether the in vitro conditions mimic MTB infection in vivo.
In summary, Treg cells play a role in masking LTBI by suppressing the specific MTB
immune response through altering IFN- and IL-10 production.
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4. Serological diagnosis of tuberculosis
The diagnosis of tuberculosis infection remain unchanged or with very limited progress for
many decades. Until recent years, diagnosis of tuberculosis primarily depends on traditional
sputum microscopy for acid fast bacilli (AFB) in low and middle income countries where the
disease is heavily concentrated, although the sensitivity of the method is variable (20~60%)
(Steingart et al., 2006). In HIV/AIDS infection, frequent occurrence of non-cavitary
pulmonary lesion can cause sputum negative tuberculosis disease. Extra-pulmonary
involvement is 10-20% of all tuberculosis case and can occur relatively frequently in children
than adults and in HIV/AIDS infection than healthy. Since enhancement of B cell immunity
and production of antibody along with protective cell-mediated immunity may play an
important role in the immunopathogenesis of tuberculosis, detection of specific antibodies
against various mycobacterial derived antigens could also play a significant role in the
diagnosis of tuberculosis. The value of various mycobacterial native or recombinant protein,
lipid or different combinations of purified antigens or commercially available kits as a
potential candidate of active tuberculosis sero-marker was evaluated by the ELISA method
in many attempts (Abebe et al., 2007; Verma & Jain, 2007; Steingart et al., 2009).
Development of a serological test with sufficient diagnostic efficacy for tuberculosis
diagnosis could be very much useful tool in resource-limited countries, as the procedures
are simple, relatively cost effective and can be performed rapidly.
Characteristics of commonly used antigens have been listed in the following table (Table 2).
Until recently, various mycobacterial culture filtrate and surface exposed proteins including
38kDa, Ag85B, MPT51, Ag60 antigens, malate synthase, heat shock protein, RD1 antigens
were investigated to determine their diagnostic efficacy.
Target
antigen
Type of antigen
Mycobacterial
location/Rv region
LAM
Lipoglycan
Cell wall
TDM
Glycolipid
Cell wall
DAT
TAT
SL-I
38kDa
Ag85B
Malate
synthase
MPT51
ESAT-6
CFP-10
Antigen 60
Glycolipid
Glycolipid
Glycolipid
Protein
Protein
Cell wall
Cell wall
Cell wall
Rv0934
Rv1886c
Immunomodulation,
Anti-inflammatory
Immunomodulation,
Enhance inflammation and
granuloma formation
Immunmodulation
Immunomodulation
Related to MTB virulence
Immunogenic protein
Immunogenic protein
Protein
Rv1837c
Immunogenic protein
Protein
Protein
Protein
Glycopeptidolipid
3803c
3875
3874
Immunogenic protein
Immunogenic protein
Immunogenic protein
Immunomodulation
Biological effects
Table 2. Characteristic of mycobacterial antigens commonly assessed for the serodiagnosis
of tuberculosis.
LAM: lipoarabinomannan; TDM: trehalose 6,6 dimycolate; DAT: 2,3-diacyl trehalose; TAT:
́́
2,3,6-triacyl trehalose; SL-I: 2,3,6,6-tetraacyl trehalose 2́’-sulphate
(Sulfolipid-I); Ag85B:
Antigen85B; ESAT-6: early secreted antigenic target-6; CFP-10: culture filtrate protein-10
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4.1 Serodiagnostic markers
38kDa antigen: The 38-kDa antigen (also known as Antigen 5) is a major lipoprotein antigen
of M. tuberculosis. As reviewed by Steigart et. al in a meta-analysis, it yielded a sensitivity
of 47% and a specificity of 94% in smear positive tuberculosis patients. The increased
sensitivity is found to be associated with smear-positive than negative tuberculosis
(Wilkinsonson et al. 1997; Julian et al., 2000). Use of native or recombinant protein did not
show any difference in term of diagnostic efficacy. But, the sensitivity of IgG detection was
relatively higher than that of IgA against 38 kDa antigen (Verma & Jain, 2007). Several
commercially available antibody detection kits were also developed using this antigen
including Pathozyme Myco (LAM+38kDa), Pathozyme TB complex plus (38kDa+16kDa).
The sensitivity varies in both Pathozyme Myco (21-46%) and Pathozyme TB complex plus
(29-76%). However, the tests are highly specific (94-100%) for tuberculosis diagnosis
(Steingart et al., 2007). The sensitivity was less than 70% and the specificity varies from 70%
to 94.9% in different studies for the diagnosis of smear-positive tuberculosis patients coinfected with HIV (Abebe et al., 2007). The sensitivity of Pathozyme Myco and Pathozyme
TB complex plus varies from 11% to 51% respectively, although the specificities were more
than 90% by both the kits for the diagnosis of extra-pulmonary tuberculosis (Steigart et. al.,
2007).
Antigen 85B: It is a member of a family of Ag85 protein complex (Ag85A, Ag85B and
Ag85C). It is a major fraction of secreted proteins in the MTB culture filtrate and cell wall. A
relatively higher sensitivity in HIV-positive tuberculosis patients (62%) than HIV-negative
tuberculosis patients (53%) with a high specificity (>95%) in both groups were reported for
Ag85B (Steingart et al., 2009). MPT51 is an antigen also related to the protein family of Ag85
complex. MPT51 provided comparable diagnostic efficacy in both HIV-negative (sensitivity:
59%) and HIV-positive tuberculosis patients (sensitivity: 58%) and the specificities of 94%
and 97% respectively (Steingart et al., 2009).
ESAT-6 and CFP-10: These are two low molecular weight secreted proteins, encoded within
the RD1 region of M. tuberculosis and highly associated with the virulence of the organism.
It is known to induce strong cell mediated immune response. Although the use of ESAT-6
and CFP-10 in IFN- based immunological methods for tuberculosis is widely acceptable for
the diagnosis of active and latent tuberculosis infection, the potential use of this antigens in
antibody-based diagnostic methods were also evaluated. Association of antibody responses
against ESAT-6 was described to be related with inactive stage of tuberculosis (Davidow et
al., 2005; Doherty et al., 2002), although increased antibody in progressive tuberculosis was
also demonstrated in other report (Demissie et al., 2006). In addition, CFP-10 showed a
sensitivity of 48% and a specificity of 96% for the diagnosis of active pulmonary
tuberculosis. The use of CFP-10/ESAT-6 fusion antigen obtained a relatively higher
sensitivity of 60.4% and a specificity of 73.8% in HIV-seronegative tuberculosis patients (Wu
et al., 2010).
Antigen 60 (A60): It is a heat-stable component of PPD extracted from BCG that can be
recognized by the sera of tuberculosis patients (Abebe et al., 2007). Anda TB (Anda
Biologicals, Strasbourg, France), a commercially available ELISA kit was developed using
A60 and its diagnostic ability was evaluated by many investigators for the diagnosis of
pulmonary and extra-pulmonary tuberculosis. The sensitivity was variable in pulmonary
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tuberculosis (29%-85%) as well as in extra-pulmonary tuberculosis (0%-100%). However, the
specificities ranges 70%-100% for both types of tuberculosis (Steingart et al., 2007).
Non-peptidic antigens from the mycobacterial cell wall grasp the main focus of
comprehensive research for the determination of their potential role in the protective
immunity or marker of TB disease.
Lipoarabinomannan (LAM): LAM, a complex glycolipid antigen forming is a major part of
cell wall of MTB. Evaluation of anti-LAM-IgG against purified LAM from MTB for the
serodiagnosis of tuberculosis was reported to have good diagnostic ability (sensitivity: 91%,
specificity: 72%) in detection of both pulmonary, pleural and miliary tuberculosis as well as
tubercular lymphadenitis (Sada et al., 1990). However, lower rate of sensitivity (50.5%) and
comparable specificity (78.3%) for the diagnosis of pulmonary tuberculosis patients were
reported by Tessema et al. (2002). In addition, MycoDot, (Genelabs, Switzerland), a
commercially available kit for the detection of antibodies against MTB specific LAM was
also evaluated in many reports (Steingart et al., 2007; Verma & Jain, 2007). Although the
specificities were high (84-100%), low rate of sensitivities (16-56%) were obtained and low
sensitivities were mostly related to HIV/TB co-infection (Verma & Jain, 2007)
DAT, TAT and SL-I: Assessment of antibody responses using DAT, TAT, and SL-I antigens
in ELISA for serodiagnosis of tuberculosis revealed variable results in terms of diagnostic
efficacy. Widely variable ranges of sensitivity of 11 to 88% by DAT antigen were reported in
several studies. A similar rate of sensitivity by MTB (44.5%) and M. fortuitum (48.6%)
infection were also demonstrated. In addition, the test sensitivities of TAT anigen also vary
from 51-93% (Julian et al., 2002). Julian et. al. reported the best performance of IgG
(Sensitivity: 81% and specificity: 77.6%) and IgA (sensitivity: 66% and specificity: 87%)
antibodies by SL-I among four trehalose contacting glycolipids (DAT, TAT, SL-I, TDM).
Although, several reasons including variation in the ELISA protocol, using of different
antigen concentrations and population from different subgroups were described as possible
reason of such variability, the effectiveness of theses antigens are still uncertain.
TDM (also known as cord factor): It composes a major part of the mycobacterial cell wall,
was identified as the most immunogenic glycolipid. Clinical evaluation of serodiagnosis of
pulmonary tuberculosis using of TDM purified from Mycobacterium tuberculosis H37Rv, in
ELISA reported its sensitivity of 81% and its specificity of 96% (Mizusawa et. al. 2008). Best
performance by TDM (sensitivity 69%, specificity: 91%) among other lipid antigens
including DAT, TAT, SL-I for the serodiagnosis of tuberculosis were also reviewed by
Steingart et al. (2009). IgG antibody against TDM can also recognize mycolic acid sub-classes
and highly active against methoxy-mycolic acid in the cord factor of M. tuberculosis than
keto-mycolic acid in M. avium complex (Pan et al., 1999). By combining TDM with more
hydrophobic glycolipids, a new tuberculous glycolipid (TBGL) antigen was designed and a
more sensitive serodiagnostic kit for TB, an anti-TBGL IgG test was developed (Kyowa
Medex Co, Japan). Anti-TBGL IgG antibody (TBGL IgG) has been proposed as a useful serodiagnostic marker of active pulmonary tuberculosis (PTB) (sensitivity: 84% and specificity:
95% in young adults) in Japan (Maekura et al., 2001). Strong association between TBGL IgG
and IgA was also revealed in active pulmonary tuberculosis cases and increased TBGL IgG
and IgA was found to be associated with CRP and cavity formation indicating their
involvement in the disease pathogenesis (Mizusawa et al., 2008). Elevated titers of TBGL IgG
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were found in aged (>40 yrs) healthy control people (17%) in Japan (Maekura et al., 2001)
and high titers of both TBGL IgG (46%) and IgA (36%) in healthy adults was also observed
in our recent study in Thailand (Siddiqi et. al.; in press). But, elevated titers of TBGL IgG are
not related to BCG vaccination (Nabeshima et al., 2005). Very recently, significant
association between the TBGL IgG and Quatiferon-TB Gold IT assay responses in diagnosis
of latent tuberculosis infection in healthy healthcare workers was also found (Siddiqi et. al.,
unpublished data) that represents enhancement of humoral immune responses along with T
cell mediated immunity in latent tuberculosis infection. Increased synthesis of TBGL IgA
titers that was related to serum IgA were observed in HIV carriers with low CD4+ T cells
counts (less than 350/µl) compared to high CD4+ T cells counts (more than 350/µl).
However, the sensitivities of TBGL IgG and IgA were very low (10% and 8% respectively)
although their specificities were more than 90% for the diagnosis of tuberculosis in pediatric
cases (Siddiqi et. al., 2009, unpublished data).
4.2 Discussion
The performance of various purified antigens and commercially available kits for the serodiagnosis of active pulmonary tuberculosis with or without associated HIV/AIDS coinfection were evaluated in many studies and were reviewed extensively (Abebe et al., 2007;
Steingart et al., 2006, 2007, 2009; Varma & Jain, 2007). Protein antigens were reported to have
high specificity (>95%) than lipid antigens. In relation to use of single antigen, relatively
higher sensitivities can be achieved by using multiple antigens. Cord factor (TDM), among
the lipid antigens had the best overall performance. In addition, higher rate of sensitivity
can be obtained by evaluation of IgG and/or IgA than IgM. Maes and colleagues has been
conceptualized the human immune responses against mycobacteria into four different
stages based on BCG vaccination and tuberculosis disease and treatment, initiated from
innate response followed by intermingled innate and adaptive response against low
molecular weight oligopeptiic and nonpeptidic, as muramyldipeptide and trehalose 6,6
dimycolate and high molecular weight nonpeptidic antigens such as lipoarabinomannan.
The final response is directed against protein antigens (Maes et al., 1999). Although it is not
clearly understood, enhancement of humoral immune response can be dependent on
disease pathogenesis and different stage of infection can influence different subclasses of
immunoglobulins. Enhanced IgM expression can usually occur in the early stage of infection
that can subsequently be diminished on progression of disease. Therefore, detection of IgM
may show limited value in the sero-diagnostic assay. The reason of low sensitivity of IgA
antibody is not clear. It is possible that the generation of IgA antibody needs larger amounts
of antigens and related with degree of disease pathogenesis than do IgG responses and
indicate the heterogeneity of tuberculosis infection.
However, until now, any performance was not successful to show the, stable, consistent and
acceptable sero-diagnostic efficacy with a sensitivity of at least more than 85% and a
specificity of more than 95% and to replace the traditional sputum microscopy as a reliable
diagnostic tool in different groups of tuberculosis patients including HIV-positive, negative, extra-pulmonary tuberculosis or in pediatric tuberculosis detection. However,
extensive study for evaluation of humoral immune responses in different stages of
tuberculosis infection and disease and their association with the disease pathogenesis
should be consider to clarify the variable antibody responses against different antigens. As
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most of the investigations for the determination of sero-diagnostic ability of various
antigens were carried out in tuberculosis endemic countries, determination of antibody
responses in latent tuberculosis infection could be helpful to some extent for explaining the
reason of low specificities and the possibility of influence by tuberculosis endemicity in such
countries. The immune response in HIV/AIDS patients co-infected with tuberculosis is
more complex than single infection. Antibodies against several single or multiple antigens
were detected in HIV/AIDS patients with active tuberculosis and even months to year’s
prior development of tuberculosis related symptoms in some prospective studies. More
investigation with diverse antigens for the sero-diagnosis of subclinical and active
tuberculosis particularly sputum-negative and extra-pulmonary tuberculosis especially in
those with HIV/AIDS co-infection and in pediatric cases is an urgent necessity.
It is generally believed that, T helper immunity and elaboration of IFN- offer vital role in
protective immunity to clear or containment of the intracellular MTB infection. However,
BCG-induced antibodies was shown to potentiate IFN- production by mycobacteriumspecific CD4+ T cells and also can cause enhancement of mycobacterial phagocytosis
probably by inducing opsonization by antibodies and that might be related to mucosal
immunity (Abebe & Bjune, 2009). Protective role of antibodies against several TB antigens in
mice model was also review by Glatman-Freedman ( 2009). Therefore, antibody-mediated
immunity against diverse mycobacterial antigens in synergy with cell mediated immunity
can play a vital role in the protection and immunopathogenesis of tuberculosis infection and
disease. Frequent detection of antibodies in latent or progressive stages of latent to active
tuberculosis and their relation to immune responses especially with mucosal immunity
needs to be clarified further.
5. Acknowledgment
This work is supported the Scientific Research Expenses for Health and Welfare program
from the Ministry of Health, Labour and Welfare, Japan (TH) and Science and Technology
Research Partnership for Sustainable Development from Japan Science and Technology
Agency, Japan (YS). This work was supported by collaborative funding from the Research
Centre for Zoonosis Control, Hokkaido University. We are grateful to prof. Ishii N. (Tohoku
University) and Dr. Mousavi S.F. (Pasteur Institute of Iran) for the help with Treg depletion
experiment. We would like to thank to Mr. Brent Bell for reading the manuscript.
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Understanding Tuberculosis - Global Experiences and Innovative
Approaches to the Diagnosis
Edited by Dr. Pere-Joan Cardona
ISBN 978-953-307-938-7
Hard cover, 552 pages
Publisher InTech
Published online 15, February, 2012
Published in print edition February, 2012
Mycobacterium tuberculosis is a disease that is transmitted through aerosol. This is the reason why it is
estimated that a third of humankind is already infected by Mycobacterium tuberculosis. The vast majority of the
infected do not know about their status. Mycobacterium tuberculosis is a silent pathogen, causing no
symptomatology at all during the infection. In addition, infected people cannot cause further infections.
Unfortunately, an estimated 10 per cent of the infected population has the probability to develop the disease,
making it very difficult to eradicate. Once in this stage, the bacilli can be transmitted to other persons and the
development of clinical symptoms is very progressive. Therefore the diagnosis, especially the discrimination
between infection and disease, is a real challenge. In this book, we present the experience of worldwide
specialists on the diagnosis, along with its lights and shadows.
How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following:
Beata Shiratori, Hiroki Saitoh, Umme Ruman Siddiqi, Jingge Zhao, Haorile Chagan-Yasutan, Motoki Usuzawa,
Chie Nakajima, Yasuhiko Suzuki and Toshio Hattori (2012). Immunological Diagnosis of Active and Latent TB,
Understanding Tuberculosis - Global Experiences and Innovative Approaches to the Diagnosis, Dr. Pere-Joan
Cardona (Ed.), ISBN: 978-953-307-938-7, InTech, Available from:
http://www.intechopen.com/books/understanding-tuberculosis-global-experiences-and-innovative-approachesto-the-diagnosis/immunological-diagnosis-of-active-and-latent-tb
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